Japanese encephalitis (JE) is the most important viral encephalitis in the world. It is widespread throughout Asia and is spreading beyond its traditional boundaries. There is no specific treatment for JE. Currently, 3 kinds of JE vaccine are in use in different countries, but only one is available internationally, a mouse-brain-derived inactivated vaccine. Although, this vaccine has been effective in reducing the incidence of JE, it is relatively expensive and has been linked to severe allergic and neurological reactions. Other 2 JE vaccines are only used in China, due to regulatory concern. Vaccine development for JE is a high priority on the list of World Health Organization (WHO). To date, no study has employed a replication-incompetent JEV virion as immunogens to stimulate effective immunity. We have recently developed a highly efficient packaging cell line for DNE2/AC replicon. Passaging of the pseudoinfectious virus-like particles (PVLP) on BHK-21 cell illustrated the complete absence of any infectious virus, even after 6 passages on packaging cells. Thus, toward the overall goal of developing a safer, more effective, and less costly JE vaccine, we have constructed a full-length infectious cDNA clone for JE virus (JEV) SA14-14-2 strain. Based upon these highly promising results, the Specific Aims of this Phase I SBIR proposal are: 1) construction of JEV/AC replicon for JEV SA14-14-2 strain; 2) development of stable packaging cell lines providing JE structural protein C in trains; 3) harvest of JEV PVLPs from infected packaging cell line, and analyzing the stability of the packaging cell lines and PVLP during passages; 4) preliminary analysis of the immunogenicity of the proposed vaccine in mice. Successful completion of this program will enable SBIR Phase II research including development of a cost-effective GMP process for PVLP production at industrial scale, titration of the determinants as immunogens in vivo, assessment of both the humoral and cellular responses to the PVLP, and a pre-clinical animal study of immunity to JE infection. [unreadable] Pseudoinfectious virus-like particle as vaccine candidate is inherent optimal combination of safety and efficacy. Low cost production is critical for practical use. In this proposal, we are going to develop a high efficient packaging system that can be used for large scale production. [unreadable] [unreadable] [unreadable]